Understanding the immune responses HIV envelope glycoproteins elicit during viral infections is important in designing effective prophylactic vaccines or improved diagnostic reagents. We discovered that the gp120 envelope proteins of R5 HIV-1 isolates exhibit higher immunoreactivity in standard Western blot (WB) analyses than X4 or X4/R5 dual tropic viral strains. Complex tertiary interdomain interactions renders the X4 and X4/R5 gp120s used for immunoblotting resistant to denaturation, thereby limiting the accessibility of serum antibodies to WB immunoreactive determinants. Supplementing diagnostic kits with purified R5 gp120 could improve their sensitivity and facilitate earlier diagnosis of viral infections.